Terpenoids are a group of substances including natural substances or related compounds which are structurally derived from isoprene. They differ from similar terpenes in that they contain functional groups, whereas terpenes are pure hydrocarbons.
Cyclic triterpenes are a diverse group of secondary metabolites which result from the metabolic pathway of squalene. They occur principally in plants and are of considerable interest to the pharmaceutical and food industries on account of their biological (inter alia antifungal, antibacterial, anti-inflammatory, antioxidative, antiviral and anti-tumoral) activities. Pentacyclic triterpenes constitute a particularly relevant sub-group of cyclic triterpenes. The basic structures of pentacyclic triterpenes consist of 5-ring systems with different substitution patterns of the methyl groups; in this case the rings A to D are 6-membered and ring E is five- or six-membered.
Nowadays, triterpenoids are generally obtained from higher plants by complex extraction processes (WO 2011/074766 A2, WO 2011/074766 A3R4, Muffler et al., 2011). However, in this resource they are only present in very small amounts, so that in the past it was hardly possible to commercialize and industrialize these substances (Madsen et al., 2011; Fukushima et al., 2011).
Furthermore, the chemical synthesis of biologically active triterpenes is likewise not economically viable and lasting on account of the complex structures.
The microbial production of triterpenes or triterpenoids is not yet established. It has already been shown (Moses et al., 2013) that the synthesis of pentacyclic triterpenoids in Saccharomyces cerevisiae (S. cerevisiae) is possible in principle after heterologous expression of corresponding genes (Moses et al. 2013).
Some genes which catalyze the synthesis of triterpenes are already known (Fukushima et al., 2011, Philips et al., 2006, Wang et al., 2011). These genes code for enzymes which catalyze the synthesis of for example cycloartenol or lanosterol (non-pentacyclic triterpenes) or lupeol or β-amyrin (pentacyclic triterpenes), but also corresponding secondary products (Huang et al., 2012, Kirby et al., 2008).
Lupeol or α/β-amyrin constitute the most important starting substances for the biosynthesis of a plurality of pentacyclic triterpenes. These compounds include, for example, betulinic acid, ursolic acid and oleanolic acid, which are of considerable interest to the pharmaceutical and food industries on account of their inter alia antibacterial, antiviral, anti-inflammatory and anti-tumoral activities (Fukushima et al., 2011; Saleem et al., 2009; Siddique et al., 2011; Holanda et al., 2008; Melo et al., 2011; Chintharlapalli et al., 2011; Shanmugam et al., 2011, Suzuki et al., 2002).
The production of the triterpenoid lupeol and betulinic acid in yeast is described in CN102433347. S. cerevisiae strains are known from Fukushima et al., 2011, which produce oleanolic acid, ursolic acid or betulinic acid. S. cerevisiae strains are known from Huang et al., 2012, which produce between 0.045 and 0.1 mg/L oleanolic acid, ursolic acid or betulinic acid. Dai et al., 2013, describe the synthesis of the triterpenoid protopanaxadiol with the overexpression of the tHMG1 gene as well as a NADPH-cytochrome P450 reductase in S. cerevisiae. In order to increase squalene and 2,3-oxidosqualene, the following genes were overexpressed: tHGMG1, ERG20, ERG9 and ERG1. In Fukushima et al., 2013, the synthesis of the pentacyclic triterpenoids soyasapogenol B, gypsogenic acid and 4-epi-hederagenin in S. cerevisiae is described. In Kunii et al., 2012, the oxidation of beta-amyrin to 12,13-epoxy in S. cerevisiae is described. In Seki et al., 2008, the oxidation of beta-amyrin to 11-oxobeta-amyrin in S. cerevisiae with a yield of 1.6 mg/L is described.
It is known from Wang et al., 2011, that approximately 50 oxidosqualene cyclases from plants, which catalyze the cyclization of 2,3-oxidosqualene in different triterpene alcohols, were cloned and characterized by means of heterologous gene expression in yeast. From Kirby et al., 2008, an S. cerevisiae strain is known which expresses a beta-amyrin synthase of the plant Artemisia annua and produces 6 mg/L of the triterpenoid beta-amyrin and also expresses the tHMG1 gene.
It is known that the overexpression of the HMG-CoA reductase in yeast leads to the enrichment of the triterpene squalene (Polakowski et al., 1998). Furthermore, overexpressed genes from the ergosterol biosynthesis lead to the accumulation of sterols in the yeast Saccharomyces cerevisiae (Veen et al., 2003).
Li et al., 2013, constructed S. cerevisiae strains which produce the pentacyclic triterpenoid betulinic acid in different quantities (0.01-1.92 mg L−1 OD−1). However, the achieved quantities are in no way sufficient for production on an industrial scale.
Phytochemicals such as terpenes and sterols currently make up a large proportion of active substances obtained from plants. The annual turnover is approximately 12.4 billion USO (Raskin et al., 2002). In this case there is great interest in betulinic acid, which has proved successful as an inhibitor of melanoma and other cancer cells (Pisha et al., 1995; Sunder et al., 2000). An equally important role is played by several derivatives of betulinic acid which are currently at the center of various clinical studies for the treatment of the HIV virus. The great interest in betulinic acid is accounted for above all by the therapeutically application of betulinic acid and betulinic acid derivatives against cancer or HIV (DE69908397T2, DE1971376884, DE19713768A 1, DE69634951 T2, DE69633398T2).
In addition to the outdated and inefficient synthetic production (Ruzicka et al., 1938), nowadays betulinic acid is obtained by extraction from higher plants, for example from the bark of Picramnia pentandra (Ruzicka et al., 1938), Arbutus menziesii (Robinson et al., 1970) or Ziziphus mauritiana (Pisha et al., 1995) and in particular Platanus occidentalis. In this case in spite of continuous improvement of the extraction process large quantities of organic solvent are consumed. In this connection one of the most recent processes is described in US2007/0149490A1, in which the betulinic acid is obtained from the bark of, the plane tree by means of chemical extraction. It can be seen from the document that large quantities of organic solvents as well as large quantities of energy are consumed in order to obtain betulinic acid.
Furthermore, the pentacyclic triterpenes and/or triterpenoids in plant resources only occur in the form of mixtures, so that the purification of individual components is very complex.
In order to estimate the future annual world requirement for betulinic acid a comparison may be made with taxol, which is used in cancer therapy. Betulinic acid also has, in addition to other applications (anti-inflammatory, antibacterial, antiviral), the potential for use in cancer therapy. The annual world requirement for taxol is currently approximately 1000 kg (Cameron et al., 2002). However, it must be noted that, by comparison with betulinic acid, taxol is used in much smaller doses for therapy.
Thus the disadvantages of the prior art reside above all in the fact that large quantities of solvent and energy are required by the previously available industrial processes for production of triterpenes and triterpenoids, in particular betulinic acid. Moreover, these are particularly time-consuming and expensive production processes. The described processes for microbial production currently do not achieve a yield which enables production on an industrial scale.
Therefore, the object of the invention was to provide a strain and a method for microbial production of pentacyclic triterpenoids.